PROJECT SUMMARY Novel Antitrypanosomal Alkaloids for Chagas Disease Chagas disease (American trypanosomiasis) is a WHO Neglected Tropical Disease and CDC Neglected Parasitic Infection estimated to infect 8 million people worldwide, mainly in Latin America, with increasing incidence in the U.S. (?300K). It is caused by the protozoan Trypanosoma cruzi, which is transmitted primarily via the feces of triatomine bugs, blood-sucking insects that feed on humans and animals, as well as via blood transfusions and from infected mother to fetus in utero. Clinical development of Chagas disease involves two distinct stages: an acute phase, usually asymptomatic, followed by a chronic phase, in which ?30?40% of infected individuals develop life-threatening, irreversible cardiomyopathy and/or digestive megasyndromes, often many years after the initial infection. The only available therapeutics, benznidazole and nifurtimox, were developed decades ago and suffer serious limitations, including poor efficacy in the chronic phase of the disease, long treatment regimens (60?90 days), significant side effects that lead to treatment discontinuation, contraindication during pregnancy leading to congenital infections, and intrinsic drug resistance in some strains. Accordingly, there is an urgent need for new, more effective, less toxic drugs to treat Chagas disease. To address this problem, the labs of Derek Tan (MSKCC) and James McKerrow (UCSD) have recently identified two novel antitrypanosomal alkaloid lead compounds with nanomolar in vitro activity against intracellular amastigotes, no cytotoxicity against mammalian host cells, acceptable pharmacological properties, and promising in vivo efficacy in a mouse model of acute Chagas disease. Based on these strong preliminary results, we propose herein to develop optimized lead compounds with potent in vitro antitrypanosomal activity, low host cell cytotoxicity, suitable pharmacological properties, clean toxicological profiles, and potent in vivo efficacy. We will also seek to determine mechanisms of action of the optimized leads. Our multidisciplinary, collaborative team has comprehensive expertise in organic synthesis, medicinal chemistry, chemical biology, parasitology, pharmacology, toxicology, and mouse models of Chagas disease. We will pursue this work through two specific aims: (1) We will design and synthesize analogues of the lead compounds to elucidate structure?activity relationships and address any pharmacological liabilities. (2) We will evaluate analogues for in vitro antitrypanosomal activity, mammalian cell cytotoxicity, and pharmacological and toxicological properties; in vivo pharmacokinetics, toxicology, and efficacy in a mouse model of acute Chagas disease; and mechanisms of action. Our overall goal is to develop one or more optimized lead compounds for further preclinical and ultimately clinical development toward new drugs to treat Chagas disease.